The ocular lens is a unique structure exquisitely designed to focus light onto the retina, a process that requires substantial shape changes of the lens according to the distance of the eye from the object it is focusing on. We are interested in the structure and function of membrane proteins in the lens, which play crucial roles in maintaining lens homeostasis and transparency. Membrane channels and transporters are the basis for a microcirculation system that supplies deeper-lying fiber cells with nutrients and clears them of waste products. Membrane proteins also mediate the tight packing of the fiber cells, thus helping to avoid light scattering by the lens tissue. This proposal focuses on the two major membrane proteins in lens fiber cells, the tetraspanin MP20 and the water channel AQP0. Using two-dimensional (2D) AQP0 crystals, we are also working towards understanding the general principles that underlie non-specific interactions of lipids with membrane proteins. In the previous funding period, we have produced a 1.9 [unreadable] density map of double-layered 2D crystals of AQP0, which resolved the lipid molecules surrounding the protein. Specific Aim 1 of this proposal is to further study non-specific lipid-protein interactions. We will expand the electron crystallographic data set to model alternative conformations of the lipids surrounding AQP0. We will also attempt to extend the resolution of the density map to 1.5 [unreadable], which may allow us to visualize charges of residues in AQP0. We are particularly interested in the charge state of histidines 44 and 60 at different pH values, as these two residues have been implicated in the pH regulation of water conduction by AQP0. In addition, we will visualize AQP0 in 2D crystals grown using lipids with different acyl chains and head groups. These experiments will allow us to systematically investigate the lipid characteristics that define non-specific lipid-protein interactions and to understand how lipids and proteins adapt to each other. We also obtained hexagonal 2D crystals of AQP0, and Specific Aim 2 of this proposal is to use a variety of structural and biophysical methods to elucidate how a protein designed to form tetramers assembling into square arrays can form hexagonal 2D crystals. Structural information on MP20 as well as other tetraspanins is still sparse. Specific Aim 3 is thus to determine the structure of MP20 primarily by electron microscopy but also pursuing X-ray crystallography. The structural information obtained for MP20 may be useful to model the structure of other members of the tetraspanin family, which are important in many cellular processes, such as cell adhesion, proliferation, activation, migration, and apoptosis. In Specific Aim 4 we will characterize the function of MP20 as a cell adhesion molecule by studying its interaction with galectin-3, a prominent adhesion modulator. We will also determine whether MP20 can bind to lens-specific integrins, as many tetraspanins are known to interact with integrins, especially if these contain a ?1 subunit. PUBLIC HEALTH RELEVANCE The importance of aquaporin-0 and MP20 for proper lens function is illustrated by the fact that mutations in either one of these two membrane proteins lead to the formation of cataracts. A wealth of biochemical and biophysical studies has also established the importance of protein-lipid interactions for the assembly, stability, and function of membrane proteins. Almost nothing is known about how lipids affect the structure and function of membrane proteins, whose dysfunction are the cause of a large number of human disorders.